A new paper published today in Molecular Cell by Avacta collaborators at the MRC Laboratory of Molecular Biology highlights the impact of Affimer reagents in the research of atypical ubiquitination. Dr David Komander’s research group have used Affimer binders specific for K6 and K33/K11 ubiquitin linkages to develop a better understanding of these atypical linkages both in vitro and in vivo.
Although ubiquitination plays an important role in many cellular processes, little is known about the biological functions of atypical ubiquitin linkages (M1, K6, K11, K27, K29 and K33) due to the lack of available tools to pursue this research. Ubiquitin was initially discovered in the late 1970s, and later found to form 8 different ubiquitin linkages, each of which potentially serves different signalling roles within the cell. While the most common K48 and K63 ubiquitin linkages have been investigated extensively, the high level of conservation in this pathway has meant that in all this time no effective antibodies could be generated for the atypical ubiquitin linkages, preventing research in this area. Turning our Affimer technology to this problem allowed us to develop linkage-specific Affimer binders to both K6 and K33/K11 linkages.
David Komander’s lab in Cambridge have characterised these binders to demonstrate their performance as specific, high affinity reagents across a number of applications from pull-downs and western blots to mass spectrometry and confocal microscopy. In the current publication, they detail the functionality of these ubiquitin linkage-specific Affimer proteins and use these binders to identify E3 ubiquitin ligases that assemble K6 chains. They present the structure of the K6 and K33 diubiquitin molecules bound to their cognate Affimer binders, demonstrating how the Affimer reagents achieve high specificity to the polyubiquitin targets.
Crystal structures of the Affimer binders with their atypical diubiquitin targets showed that the Affimer proteins bound the diubiquitin as dimers, using this dimerization mechanism to increase the specificity and affinity of binding. Through studying the structure of the K33 Affimer binder, the researchers were able to predict a cross-reactivity of the K33 Affimer binder to K11 linkages. As a result of the identified binding mechanism, they created dimeric fusions of the Affimer binders, thereby increasing the binding affinity to the pM range, with a low nM binding affinity of the K33 Affimer to K11 diubiquitin.
The PINK1/Parkin pathway mediates the removal of malfunctioning mitochondria to protect the cell from damage and depends upon the regulation of K6 linkages on mitochondria. Using a GFP-tagged K6 Affimer binder, the team of researchers were able to visualise this use of K6 polyubiquitin chains in mitophagy. Under situations of mitochondrial damage, the K6 ubiquitin chains localised to the cellular mitochondria. Significantly, this effect was absent in Parkin mutant cells, indicative of a role for this linkage in mitochondrial turnover. This was supported by western blots demonstrating a significant increase in K6 linkages following mitochondrial depolarisation, while the overall level of ubiquitinated proteins showed no increase.
Use of the K6 Affimer binders in pull-down experiments revealed the E3 ligase HUWE1 as a target for this atypical chain type. “We could further show that HUWE1 assembles K6 chains both in vitro and in vivo and in fact seems to be a major source of cellular K6 chains”, says Martin Michel, the first author of the study. Indeed, they could show that HUWE1 modifies Mitofusin-2 with K6 chains and that the K6 Affimer binder protected the K6 polyubiquitin chains from degradation by a deubiquitinase, whilst other ubiquitin linkages were removed from the substrate.
These exciting findings by David Komander’s group using Affimer reagents open the door to the investigation of both atypical K6 and K33 ubiquitin linkages across the biological field, which has proven previously impossible in this way, and holds potential impact for drug discovery within this developing field.
- The K6 Affimer binders used in this study can be purchased here.